Several Salmonella typhimurium strains have been used widely in what is known as the Ames test to detect mutagenic agents. In this test, bacterial strains that require histidine for growth (His-), are mutated or reverted to wild type (His+) when treated with the test mutagenic agent. Thus the presence of a mutagen is indicated by the reversion of auxotrophic strains (His-) to wild type phenotype (His+). Many such strains, e.g., TA97a, TA98, TA100, TA102 and TA104 etc., are known in the art. While these bacterial strains allow the detection of mutagens they do not provide information on which of the six possible base pair substitution mutations are caused by the mutagen.
Three systems, employing cells other than Salmonella, exist in the art for detecting each of the six possible base substitution mutations (Cupples et al., 1989; Hampsey, 1991; Schaff et al., 1990; Stambrook et al., 1988). Six strains of Escherichia coli with mutations in the lacZ gene have been used by Cupples et al. (1989). These were developed for detecting specific base substitution mutations based on phenotypic reversion to wild type. However, these strains have not been widely used because of the unwieldiness of the mutagenicity assays and because phenotypic reversion has not been demonstrated conclusively to result exclusively from a specific base substitution mutation.
Another system has been developed using six yeast strains capable of detecting specific base substitution mutations without DNA sequence analysis, by reversion of the mutants to the wild type phenotype (Hampsey, 1991). However, yeast are more difficult to maintain and the mutagenicity assay requires almost two weeks to complete compared to 48 h for the Salmonella assay. Similar to the E. coli system of Cupples et al. (1989), phenotypic revertants of the yeast strains have not been sequenced adequately to confirm reversion via a single base substitution mutation. Thus, the possibility of other mechanisms of reversion, e.g., by missense suppression cannot be eliminated as causative of the observed phenotypic reversion.
Tester mammalian cells capable of detecting specific point mutations have also been reported (Schaff et al. 1990; Stambrook et al, 1988; U.S. Pat. No. 4,792,520). These references are essentially by the same authors and disclose similar subject matter. The references relate to a cell assay for specific base substitutions and frame shift mutations using non-reverting mammalian tester cells engineered to contain a mutated mouse adenine phosphoribosyl-transferase (APRT) gene. Specific point mutations are tested by treating tester cells (APRT-) with a mutagen and observing phenotypic reversion to APRT+. However, mammalian cells are much more expensive and too cumbersome for routine screening of mutagenic agents. The method of producing the mammalian tester cells relies on targeting the nucleotide sequence at one of the intron/exon junctions in the APRT gene. This method is clearly not relevant to prokaryotic cells like Salmonella typhimurium, as splicing of RNA is not characteristic of prokaryotic cells.